Peste des Petits Ruminants (PPR) is a highly infectious disease caused by a virus of the Morbillivirus genus. The current PPR eradication effort relies mainly on the implementation of massive vaccination campaigns. One of the most widely used PPR vaccines is the Nigeria 75/1 strain obtained after attenuation by 75 serial passages of the wild type isolate in cell cultures. Here we use high throughput deep sequencing of the historical passages that led to the Nigeria 75/1 attenuated strain to understand the evolution of PPRV attenuation and to assess the risk of reversal in different cell types. Comparison of the consensus sequences of the wild type and vaccine strains showed that only 18 fixed mutations separate the two strains. At the earliest attenuation passage at our disposal (passage 47), 12 out of the 18 mutations were already present at a frequency of 100%. Low-frequency variants were identified along the genome in all passages. Sequencing of passages after the vaccine strain showed evidence of genetic drift during cell passages, especially in cells expressing the SLAM receptor targeted by PPRV. However, 15 out of the 18 mutations related to attenuation remained fixed in the population. In vitro experiments suggest that one mutation in the leader region of the PPRV genome affects virus replication. Our results suggest that only a few mutations can have a serious impact on the pathogenicity of PPRV. Risk of reversion to virulence of the attenuated PPRV strain Nigeria 75/1 during serial passages in cell cultures seems low but limiting the number of passages during vaccine production is recommended.

Peste des petits ruminants (PPR) is an acute disease of small ruminants caused by a morbillivirus. Clinical observation of the disease in the field revealed that several species of small ruminants are affected to varying degrees. This difference in disease-related effects could depend either on the host or on the virulence of the virus strain. A previous study highlighted the difference in virulence between two strains of PPRV used to infect Saanen goats. For this breed, PPRV Morocco 2008 strain (MA08) was highly virulent while PPRV Côte d’Ivoire 1989 (IC89) strain induced mild disease. Experimental studies generally based on healthy and young animals do not permit exploration of the natural variability of the host susceptibility to PPRV. Therefore, building on the previous study on Saanen goats, the current study focussed on this breed of goat and used commercially available animals with an unknown history of infection with other pathogens. Results confirmed the previous disease pattern for PPRV IC89 and MA08 strains. Viral RNA detection, macroscopic and histological lesions were stronger for the highly virulent MA08 strain. We show here for the first time that viral RNA can be detected in the tissues of vaccinated animals. Viral RNA was also detected for the first time in serum samples, which is in agreement with the role of circulating immune cells in transporting the virus into host target organs. Thus, this study provides insight into the pathogenesis of strains of different virulence of PPRV and will help to better understand the onset of the disease.

Peste des petits ruminants (PPR) is an acute transboundary infectious viral disease of small ruminants, mainly sheep and goats. Host susceptibility varies considerably depending on the PPR virus (PPRV) strain, the host species and breed. The effect of strains with different levels of virulence on the modulation of the immune system has not been thoroughly compared in an experimental setting so far. In this study, we used a multi-omics approach to investigate the host cellular factors involved in different infection phenotypes. Peripheral blood mononuclear cells (PBMCs) from Saanen goats were activated with a T-cell mitogen and infected with PPRV strains of different virulence: Morocco 2008 (high virulence), Ivory Coast 1989 (low virulence) and Nigeria 75/1 (live attenuated vaccine strain). Our results showed that the highly virulent strain replicated better than the other two in PBMCs and rapidly induced cell death and a stronger inhibition of lymphocyte proliferation. However, all the strains affected lymphocyte proliferation and induced upregulation of key antiviral genes and proteins, meaning a classical antiviral response is orchestrated regardless of the virulence of the PPRV strain. On the other hand, the highly virulent strain induced stronger inflammatory responses and activated more genes related to lymphocyte migration and recruitment, and inflammatory processes. Both transcriptomic and proteomic approaches were successful in detecting viral and antiviral effectors under all conditions. The present work identified key immunological factors related to PPRV virulence in vitro.

Using a systems immunology approach, this study comprehensively explored the immunopathogenesis of peste des petits ruminants (PPR) focussing on strain-dependent differences in virulence. Saanen goats were infected either with the highly virulent (Morocco 2008 [MA08]) or the low-virulent (Ivory Coast 1989 [IC89]) strain of the PPR virus (PPRV). As expected, MA08-infected goats exhibited higher clinical scores, pronounced lymphocyte depletion, and lesions affecting mucosal and lymphoid tissues. CD4 T cells were more affected in terms of depletion and infection in peripheral blood. Transcriptional analyses of the blood and lymphoid tissue demonstrated activation of interferon type I (IFN-I) responses at 3 days post-infection (dpi) only with MA08, but comparable IFN-I expression levels with MA08 and IC89 at 6 dpi. MA08 strain induced strong inflammatory and myeloid cell-related transcriptional responses observed in tonsils but not in mesenteric lymph node. This inflammatory response in the tonsils was associated with an extensive damage and infection of the tonsillar epithelium in the crypts, pointing to a barrier defect as a possible cause of inflammation. An early and prominent downregulation of cell cycle gene networks was observed in all compartments analyzed in MA08-infected animals. This effect can be interpreted as suppressed lymphocyte proliferation that may cause immunosuppression during the first week following MA08 infection. A proteome analysis confirmed synthesis of IFN-I response proteins during infection with both strains, but only MA08 strain additionally upregulated ribosomal and inflammation-related proteins. In conclusion, the present comprehensive investigation delineates strain-dependent differences in early immunopathological processes associated with severe inflammation disease and a blunted lymphocyte proliferation. IMPORTANCE: Field observations show that the severity of PPR is highly dependent on the viral (PPRV) strains and the host infected, but the mechanisms behind these variations are not well understood. Here we compare immune response in Saanen goats infected with high (MA08) and low (IC89) virulent PPRV strains. Analyses revealed a differential immune response: early activation of IFN-I responses only with MA08 but comparable IFN-I expression levels with MA08 and IC89 at later stages. Additionally, MA08 strain triggered inflammatory and myeloid cell-related responses in the tonsils and marked suppression of lymphocyte proliferation evidenced by cell cycle arrest. CD4 T cells were found to be most affected in terms of depletion in the peripheral blood. Massive infection of the tonsils seems to induce epithelial lesions that promote the inflammatory responses. These results underscore the need to understand strain-specific differences for PPR surveillance and control.

Animal African trypanosomosis (AAT), caused by protist parasites of the genus Trypanosoma , puts upward of a million head of livestock at risk across 37 countries in Africa. The economic impact of AAT and the presence of human-infectious trypanosomes in animals place a clear importance on improving diagnostics for animal trypanosomes to map the distribution of the veterinary parasites and identify reservoirs of human-infectious trypanosomes. We have adapted the CRISPR-based detection toolkit SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for trypanosomatid parasites responsible for AAT (SHERLOCK4AAT) including Pan-trypanosomatid, Trypanozoon , T. vivax , T. congolense , T. theileri , T. simiae, and T. suis assays. To test the applicability of this technique in the field, we analysed dried blood spots collected from 200 farm and 224 free-ranging pigs in endemic and historical human African trypanosomiasis foci in Guinea and Côte d’Ivoire, respectively. The results revealed that SHERLOCK4AAT can detect and discriminate between trypanosome species involved in multiple infections with a high sensitivity. 62.7% [58.1, 67.3] of pigs were found infected with at least one trypanosome species. T. brucei gambiense , a human-infectious trypanosome, was found in one animal at both sites, highlighting the risk that these animals may act as persistent reservoirs. These data suggest that, due to their proximity to humans and their attractiveness to tsetse flies, pigs could act as sentinels to monitor T. b. gambiense circulation using the SHERLOCK4AAT toolbox.

Inflammatory signal termination is critical for the maintenance of homeostasis. Cyclic dinucleotides (CDNs) are second messengers that trigger inflammatory responses through the activation of the stimulator of IFN genes (STING) signaling platform. No broad-acting direct regulator of intracellular CDNs has been identified in mammals to date. We show that the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a major DNA damage response actor, directly interacts with the intracellular 2′3′-cGAMP CDN through its kinase domain, tempering STING activation. DNA-PKcs also acts on the 3′3′-cGAMP bacterial CDN and pharmacological STING agonists, impacting their bioactivity and ability to mount optimal antiviral responses. STING agonism has been considered as a therapeutic avenue to alleviate immunosuppression in human pathologies. By uncovering DNA-PKcs as a CDN signaling modulator and CDNs as inhibitors of DNA-PKcs kinase activity, we provide critical insights into CDN regulation, with implications for the development of STING-targeting therapeutics.